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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #345581

Research Project: Ecology and Detection of Human Pathogens in the Produce Production Continuum

Location: Produce Safety and Microbiology Research

Title: Optimized co-extraction and quantification of DNA from enteric pathogens in surface water samples near produce fields in California

Author
item Cooley, Michael - Mike
item Carychao, Diana
item Gorski, Lisa

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/27/2018
Publication Date: 3/13/2018
Citation: Cooley, M.B., Carychao, D.K., Gorski, L.A. 2018. Optimized co-extraction and quantification of DNA from enteric pathogens in surface water samples near produce fields in California. Frontiers in Microbiology. 9:448. https://doi.org/10.3389/fmicb.2018.00448.
DOI: https://doi.org/10.3389/fmicb.2018.00448

Interpretive Summary: A primary cause of fresh produce contamination is contaminated streams and lakes near the farm. Measurement of bacteria level in surface water is difficult. One indirect method is PCR, but the current methods for the purification of DNA are inadequate. Reported here is a new extraction method for simultaneous purification of DNA from different bacterial types. Additionally, genes in these DNAs were quantified using a new type of PCR called droplet digital PCR. Those water samples previously found to be contaminated by standard microbiological methods were also found contaminated by this new procedure, indicating that the method is valid. Improved quantification methods with provide knowledge of where contamination risks are greatest.

Technical Abstract: Pathogen contamination of surface water is a health hazard in agricultural environments primarily due to the potential for contamination of crops. Furthermore, pathogen levels in surface water are often unreported or under reported due to difficulty with culture of the bacteria. The pathogens are often present, but require resuscitation, making quantification difficult. Frequently, this leads to the use of quantitative PCR targeted to genes unique to the pathogens. However, multiple pathogen types are commonly in the same water sample, both gram + and gram -, leading to problems with DNA extraction. With Shiga toxin-producing Escherichia coli (STEC), Salmonella enterica and Listeria monocytogenes as target, a method was optimized to co-extract all three and quantify the level of each using droplet digital PCR (ddPCR). Multiplexed target genes in STEC were virulence genes, shiga toxin 2 (stx2) and hemolysin (ehx). Likewise, multiplexed targets in Listeria and Salmonella were the virulence genes listeriolysin (hly) and invasion protein A (invA). Water samples were processed using microbiological techniques for each of the pathogens and duplicate water samples were quantified by ddPCR. A significant correlation was found between culture and ddPCR results indicating detection primarily of culturable cells by ddPCR. Average virulence gene levels were 923, 23k, 69 and 152 copies per sample for stx2, ehx, hly and invA, respectively. Additionally, stx2, ehx and inv levels were significantly correlated (P<0.05, R=0.34) with generic E. coli MPN levels in the duplicate samples. Indirect quantification with ddPCR will improve understanding of prevalence of the pathogens and may reduce risks associated with contaminated surface water.